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Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter
流式细胞术分离狼疮小鼠肾脏的树突状细胞和巨噬细胞   

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参见作者原研究论文

本实验方案简略版
The Journal of Immunology
Apr 2011

 

Abstract

Methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE are essential to understand the patho-physiology of the disease. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans.An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that lead to renal impairment in SLE and other renal inflammatory diseases.

Materials and Reagents

  1. Fetal bovine serum (FBS)
  2. Sterile PBS (Life Technologies, Invitrogen™, catalog number: 20012-027 )
  3. Ammonium chloride
  4. Collagenase Type I (CLS I) (Worthington, catalog number: 4197 , specific activity 230 U mg-1)
  5. DMEM, High glucose (Life Technologies, Gibco®, catalog number: 10313 )
  6. Paraformaldehyde (Tousimis, catalog number: 1108A )
  7. BSA (Fraction V) (Sigma-Aldrich, catalog number: A-7030 )
  8. FACS antibodies:
    1. FC block (BD Biosciences, Falcon®, catalog number: 553142 )
    2. CD11B APC (BD Biosciences, Falcon®, catalog number: 553310 )
    3. F/480 FITC  (Serotech Laboratories, catalog number: MCA497FB )
    4. Streptavidin PERCP (BD Biosciences, Falcon®, catalog number: 554064 )
    5. CD4, PE (BD Biosciences, Falcon®, catalog number: 553049 )
    6. CD5, PE (eBioscience, catalog number: 12-0051-82 )
    7. B220, PE (BD Biosciences, Falcon®, catalog number: 553090 )
    8. CD49b, PE (BD Biosciences, Falcon®, catalog number: 558759 )
    9. CD11C (Biotin, catalog number: 553800 )
    10. Streptavidin PERCP (BD Biosciences, Falcon®, catalog number: 554064)
  9. DAPI (MP Biomedicals, catalog number: 157574 )
  10. FACS staining buffer (see Recipes)

Equipment

  1. FACS Aria or similar cell sorter
  2. Bench-top refrigerated centrifuge
  3. BD cell strainer (40 nm) (BD Biosciences, Falcon®, catalog number: 352340 )
  4. Conical tube
  5. 30 ml syringe (BD Biosciences, Falcon®, catalog number: 309661 )
  6. 21G Needles (BD Biosciences, catalog number: 305165 )
  7. 26G needles (BD Biosciences, catalog number: 305111 )
  8. V bottom 96 well assay plate (Corning, Costar®, catalog number: 3897 )
  9. Glass slides frosted (Thermo Fisher Scientific, catalog number: 12-550-11 )

Procedure

  1. Procedure for harvesting the kidney from the nephritic mice for analysis of kidney infiltrates
    1. Anesthetize the mouse and perfuse with 60 ml of cold PBS over 3-5 min through the left ventricle after snipping the right atrium, and observe for pale white color change in liver and kidney. If needed, repeat perfusion with another 60 ml of cold PBS.
    2. Carefully remove and cut the kidneys into 1 to 2 mm3 pieces, excluding any adjoining renal fat.
    3. Incubate the slices in DMEM containing 2 mg/ml Collagenase Type I (Worthington) for 30 min at 37 °C (use 10 ml per two kidneys).
    4. Gently disrupt the tissue by pipetting up and down sequentially through 25 ml, 10 ml, and 5 ml pipettes to obtain a fine cell suspension.
    5. Filter the cell suspension through a BD cell strainer (40 nm) into a conical tube.
    6. Gently rub the remaining material between two glass slides, resuspend in 2 ml DMEM, filter and add to the suspension.
    7. Allow the suspension to settle briefly (3-5 min) during which most of the larger fragments settle to the bottom. Harvest the suspension excluding the bottom 200 μl containing the fragments.
    8. Examine the settled cells under microscope to see if any clumps are present. If so, resuspend the settled cells in fresh DMEM, filter and repeat step 6.
    9. Pool the suspension(s) obtained and centrifuge at 1200 rpm or 300 x g for 10 min.
    10. Decant supernatant; resuspend the pellet in 5 ml of ice cold ammonium chloride (0.17 M, pH 7.2) for 5 min on ice.
    11. Add 15 ml of DMEM. Count cells to estimate the number of total cells in the suspension. Spin at 1,200 rpm for 5 min.
    12. Resuspend cells in 1 ml of FACS buffer (3% FCS in PBS). Cells are now ready for flow cytometric analysis or further isolation procedures.

  2. Procedure for sorting renal mononuclear phagocytes (macrophages and dendritic cells) by cell sorting.
    1. Suspend the cell suspensions from both kidneys from one mouse in 2 ml of FACS buffer with Fc block and incubate on ice for 20 min.
    2. Add 7 µl of CD11c-biotin antibody to the tube and incubate for 30 min on ice.
    3. Centrifuge the cells at 1,200 rpm for 5 min and decant the supernatant.
    4. Resuspend the cell pellet in 2ml of FACS buffer containing anti-CD11b APC, F4/80 FITC, and Streptavidin PERCP. The cocktail should also include a combination of antibodies to facilitate the exclusion of unwanted cells, such as PE anti-CD3, CD5, B220, CD138 (optional) and CD49b (5-7 µl each); incubate for another 30 min.
    5. Wash the cells with 2 ml of ice cold PBS and centrifuge at 1,200 rpm for 5 min.
    6. Resuspend the cell pellet in 500 µl of FACS buffer and filter the suspension using a strainer cap FACS tube. Later, adjust the volume for the cells based on the number of events acquired per second on the cell sorter (FACS Aria IIu).
    7. Just before sorting the cells, add 2 µl of DAPI (1 µg/ml) to exclude the dead cells.
    8. Pass the cells through the sorter with a flow rate optimized to 5,000 to 7,000 events per sec and maintain the efficiency of the sort at between 75-85%.

  3. Sorting strategy
    1. Gate on lymphocytes/mononuclear cells


    2. Gate on singlets


    3. Gate on live cells (DAPI negative)


    4. Gate out unwanted cell types (B cells, plasma cells, T cells, NK cells)


    5. Separate CD11bhigh and CD11blowCD11chigh population (the former is the subject of the next step)


    6. Sort renal dendritic cells and macrophages, respectively, into FACS buffer.


    7. Process the sorted cells based on further study requirements (cell culture, RNA isolation, morphological and functional characterization, western blot analysis).

Recipes

  1. 0.17 M ammonium chloride
  2. FACS staining buffer
    PBS
    3% FBS

Acknowledgments

This work was supported by the NY SLE foundation to RB and National Institutes of Health RO1 DK085241-01 for AD.

References

  1. Bethunaickan, R., Berthier, C. C., Ramanujam, M., Sahu, R., Zhang, W., Sun, Y., Bottinger, E. P., Ivashkiv, L., Kretzler, M. and Davidson, A. (2011). A unique hybrid renal mononuclear phagocyte activation phenotype in murine systemic lupus erythematosus nephritis. J Immunol 186(8): 4994-5003.

简介

从SLE小鼠的肾中分离和表征单核吞噬细胞的方法对于了解疾病的病理生理学是必不可少的。 这些细胞的活化与小鼠中的临床疾病的发作相关,并且这些细胞的浸润与人类的不良预后相关。这些细胞的功能的分析应导致更好地理解导致肾损伤的炎症过程 在SLE和其他肾脏炎性疾病中

材料和试剂

  1. 胎牛血清(FBS)
  2. 无菌PBS(Life Technologies,Invitrogen TM,目录号:20012-027)
  3. 氯化铵
  4. 胶原酶I型(CLS I)(Worthington,目录号:4197,比活性230U mg -1
  5. DMEM,高葡萄糖(Life Technologies,Gibco ,目录号:10313)
  6. 多聚甲醛(Tousimis,目录号:1108A)
  7. BSA(级分V)(Sigma-Aldrich,目录号:A-7030)
  8. FACS抗体:
    1. FC嵌段(BD Biosciences,Falcon ,目录号:553142)
    2. CD11B APC(BD Biosciences,Falcon ,目录号:553310)
    3. F/480 FITC (Serotech Laboratories,目录号:MCA497FB)
    4. 链霉亲和素PERCP(BD Biosciences,Falcon ,目录号:554064)
    5. CD4,PE(BD Biosciences,Falcon ,目录号:553049)。
    6. CD5,PE(eBioscience,目录号:12-0051-82)
    7. B220,PE(BD Biosciences,Falcon ,目录号:553090)
    8. CD49b,PE(BD Biosciences,Falcon ,目录号:558759)
    9. CD11C(生物素,目录号:553800)
    10. 链霉亲和素PERCP(BD Biosciences,Falcon ,目录号:554064)
  9. DAPI(MP Biomedicals,目录号:157574)
  10. FACS染色缓冲液(参见配方)

设备

  1. FACS Aria或类似细胞分选机
  2. 台式冷冻离心机
  3. BD细胞过滤器(40nm)(BD Biosciences,Falcon ,目录号:352340)
  4. 圆锥管
  5. 30ml注射器(BD Biosciences,Falcon ,目录号:309661)
  6. 21G针(BD Biosciences,目录号:305165)
  7. 26G针(BD Biosciences,目录号:305111)
  8. V底96孔测定板(Corning,Costar ,目录号:3897)
  9. 玻璃载玻片(Thermo Fisher Scientific,目录号:12-550-11)

程序

  1. 从肾炎小鼠收获肾脏以分析肾脏浸润的程序
    1. 麻醉鼠标和灌注60毫升冷PBS超过3-5分钟通过左心室切断右心房后,观察肝脏和肾脏的苍白色变化。 如果需要,用另一60ml冷PBS重复灌注。
    2. 小心地将肾脏切除并切成1至2毫米 3片,不包括任何相邻的肾脏脂肪。
    3. 将切片在含有2mg/ml I型胶原酶(Worthington)的DMEM中在37℃下孵育30分钟(使用每两个肾脏10ml)。
    4. 通过向上和向下顺序通过25毫升,10毫升和5毫升吸管轻轻破坏组织,获得细胞悬浮液。
    5. 将细胞悬浮液通过BD细胞过滤器(40nm)过滤到锥形管中
    6. 轻轻擦拭两个载玻片之间的剩余材料,重悬于2ml DMEM中,过滤并加入悬浮液中。
    7. 让悬浮液短暂停留(3-5分钟),在此期间大多数较大的碎片沉降到底部。收获包含碎片的底部200μl以外的悬浮液。
    8. 在显微镜下检查沉降的细胞,以查看是否存在任何团块。如果是,将沉降的细胞重悬于新鲜DMEM中,过滤并重复步骤6
    9. 收集获得的悬浮液,并以1200rpm或300×g离心10分钟。
    10. 倾析上清液; 在冰上将沉淀重悬于5ml冰冷的氯化铵(0.17M,pH7.2)中5分钟。
    11. 加入15毫升DMEM。 计数细胞以估计悬浮液中总细胞的数量。 以1,200rpm旋转5分钟。
    12. 重悬细胞在1毫升FACS缓冲液(3%FCS的PBS)。 细胞现在可以进行流式细胞分析或进一步的分离程序

  2. 通过细胞分选分选肾单核吞噬细胞(巨噬细胞和树突状细胞)的程序。
    1. 将来自一只小鼠的两个肾脏的细胞悬浮液悬浮于2ml含Fc块的FACS缓冲液中,并在冰上孵育20分钟。
    2. 加入7微升的CD11c生物素抗体的试管,在冰上孵育30分钟
    3. 以1,200 rpm离心细胞5分钟,倾析上清液
    4. 将细胞沉淀重悬在2ml含有抗CD11b APC,F4/80 FITC和链霉抗生物素蛋白PERCP的FACS缓冲液中。混合物还应包括抗体的组合以促进排除不想要的细胞,例如PE抗CD3,CD5,B220,CD138(可选)和CD49b(各5-7μl);再孵育30分钟。
    5. 用2ml冰冷的PBS洗涤细胞,并在1,200 rpm离心5分钟
    6. 将细胞沉淀重悬于500μlFACS缓冲液中,并使用过滤帽FACS管过滤悬浮液。然后,根据在细胞分选器(FACS Aria IIu)上每秒获取的事件数量调整单元格的音量。
    7. 在分选细胞前,加入2μlDAPI(1μg/ml)以排除死细胞
    8. 使细胞通过分选机,流速优化为每秒5,000至7,000个事件,并将分选效率保持在75-85%之间。

  3. 排序策略
    1. 淋巴细胞/单核细胞上的门


    2. 单片机上的门


    3. 活细胞门(DAPI阴性)


    4. 选出不需要的细胞类型(B细胞,浆细胞,T细胞,NK细胞)


    5. 分离CD11bhigh和CD11blowCD11chigh群体(前者是下一步的主题)


    6. 将肾脏树突状细胞和巨噬细胞分别分入FACS缓冲液

    7. 基于进一步的研究要求(细胞培养,RNA分离,形态和功能表征,western印迹分析)处理分选的细胞。

食谱

  1. 0.17M氯化铵
  2. FACS染色缓冲液
    PBS
    3%FBS

致谢

这项工作得到NY SLE基金会和RB和国立卫生研究院RO1 DK085241-01 AD的支持。

参考文献

  1. Bethunaickan,R.,Berthier,CC,Ramanujam,M.,Sahu,R.,Zhang,W.,Sun,Y.,Bottinger,EP,Ivashkiv,L.,Kretzler,M.and Davidson, 。 鼠系统性红斑狼疮肾炎中的独特杂合性肾单核吞噬细胞活化表型。 J Immunol 186(8):4994-5003。
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引用:Bethunaickan, R. and Davidson, A. . (2012). Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter. Bio-protocol 2(8): e168. DOI: 10.21769/BioProtoc.168.
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